Fabrication of injectable Thermosensitive Chitosan/Pullulan Hydrogels using double cross-linking intended for cartilage tissue engineering applications

Abstract

Injectable thermosensitive chitosan/β-glycerophosphate (CS/BGP) hydrogels have been widely used in cartilage tissue engineering. These hydrogels were established via physically crosslinked networks, resulting in poor mechanical properties, thus limiting their applications. In this study, the mechanical properties of the CS/BGP hydrogels were improved by the concomitant addition of genipin (GE) and pullulan (PL) in order to obtain chemical interconnection between CS chains and semi-interpenetrating networks, respectively. The optimization of the hydrogel formulation was conducted using Box-Behnken experimental design coupled with response surface methodology. The desirable properties of the optimal formulation were defined as the maximum mechanical properties (Young’s modulus) and swelling capacity (equilibrium swelling ratio; ESR). The optimal formulation was composed of 1.05% w/v CS, 1% w/v PL, 6% w/v BGP, and 70.79 mcg/mL GE. The predicted values of Young’s moduli and ESR were in agreement with those obtained from experiments. The optimized formulation showed Young’s modulus of 92.65 ± 4.13 kPa and ESR of 3259.09% ± 58.90%. SEM analysis revealed a highly porous structure with nanofibrous networks in the optimized hydrogel. FTIR spectra indicated the chemical bonding between the functional groups of CS and GE. The applicable properties of hydrogels, namely injectability, gelation temperature, and gelation time, were evaluated. The results showed that all formulations were injectable where the infection force needed was below 30 N. Rheological analysis illustrated that the sol-gel transition of the optimized hydrogel occurred in 55.44 s at 37 °C, and the lower critical solution temperature of the hydrogel was 30.77 °C. Additionally, frequency-sweep tests and indentation tests suggested that the optimized hydrogel had a stiffer gel with higher storage modulus, compared to CS/BGP hydrogel. Furthermore, in vitro enzymatic degradation revealed that the optimized hydrogel had more durable than the hydrogels prepared by BGP or GE. In conclusion, the results in this study suggested that the injectable CS/PL hydrogels prepared by double crosslinking using BGP and GE demonstrated the sol-gel transition at physiological temperature with improved mechanical properties as well as swelling capacity, which provide the potential for use as scaffolds for cartilage tissue engineering.